Recent studies provide strong evidence that single class V myosin molecules transport vesicles and organelles processively along F-actin, taking several 36-nm steps, hand over hand, for each diffusional encounter. We demonstrated that the ATPase activity of myosin required calcium for maximal activity and showed that, in the absence of calcium, myosin V adopted a folded, inactive structure. We have now examined the structure of the inactive complex in more detail using single particle analysis of negatively stained myosin V molecules. Averaged images show considerable detail. The motor domain and lever arm of each molecule bends acutely back upon its stalk and each of the motor domains contact a lobe of the globular tail domain (GTD). The point of contact at the motor domain is a surface loop located near the nucleotide binding site that contains four negatively charged amino acids that are conserved in the myosin V family, but not necessarily in other myosin superfamily members. A truncated HMM-like fragment of myosin V lacking the distal coiled-coil domain of the stalk and the GTD has a high ATPase activity that is not affected by calcium concentration. Addition of a GST-GTD dimeric fusion protein inhibits the ATPase activity of myosin V HMM in the absence, but not the presence of calcium. Examination of the HMM-GST-GTD complex in the absence of calcium shows that the two motor domains of HMM have folded back and contacted the two GTD domains to form a structure remarkably like that seen in the intact molecule. Studies of the frequency of movement of intact myosin V and HMM in the absence of calcium in single molecule motility assays show that the former is markedly inhibited and shows many fewer movements than the latter.